The present invention relates to a method for instrument determination of a measured variable L(t) which changes with time, the intention being to determine the maximum value of L(t) in a region with a linear reaction profile, and the time as well as the size of the reaction time window being variable with the linear region and depending on the nature of the reaction and the reaction conditions. Specifically, the present invention relates to the determination of protein concentrations with the aid of light scatter, which is produced by specific antibodies, in homogeneous solutions. In particular, the invention relates to reactions which take place slowly and have a largely linear profile over a relatively long time, the rate of formation of antigen-antibody complexes in the linear section of the reaction being determined as the measured variable.
The phenomenon of light scatter on particles in a homogeneous medium is used for concentration determination, both by measuring the scattered light intensity (nephelometry) and by measuring the intensity loss of the light beam passing through the medium (turbidimetry).
The immunochemical reaction between a soluble antigen and a bivalent or polyvalent antibody leads to large groups of molecules which scatter light to a major extent. The time profile of such reactions very frequently corresponds to the general kinetic profile of successive first order reactions and has a point of inflection, so that the maximum rate of reaction does not occur until during the course of the reaction (see, for example, FIG. 1a-1c). A concentration-dependent measurement signal can be obtained in various ways from the signal-time curves in FIG. 1a-1c.
The intensity of the signal change can be increased by bonding one of the reaction partners to particles, for example in the "particle-enhanced assays" known per se to a person skilled in the art.
In the end-point method, the measurement signal is determined at a time which is so late that, on the basis of experience, it is no longer changing but no precipitation is taking place. In the "fixed time" method, the actual measurement method is the difference between two signals which are determined at times that are different but are fixed in advance.
In the kinetic "peak rate method", the maximum rate of reaction (V.sub.Max), that is to say the maximum change (.delta.) of the signal (S) per unit time (.delta.t), is determined
a) by measurements of .delta.S at sufficiently short time intervals (.delta.t) and determination of the maximum quotient .delta.S/.delta.t,
b) electronic differentiation .delta.S/.delta.t and determination of the maximum,
c) construction of the tangent to the signal/time curve and determination of the maximum gradient S=signal, t=time.
The method according to the invention can in principle be used for all determinations of a measured variable which varies with time, provided the change in the measured variable is linear only in a sub-region and is intended to be used for evaluation of the linear part.
A large number of analytes can now be quantified by direct or indirect scattered light measurement using the described methods. If one considers the dependency of a suitable measurement signal on the concentration of a reaction partner, for example the antigen, while the other reaction partner is used with a constant concentration, then, for example, it is possible in the case of immunochemical reactions to observe that the same measurement signal can be caused by both a low concentration and a high concentration of the analyte. This leads to an ambiguity in the signal concentration relationship, which is known to the person skilled in the art as the antigen excess phenomenon "high-dose hook" or Heidelberger curve. This ambiguity can in principle be observed wherever complexes of different stoichiometry are possible, depending on the excess amount of one reaction partner or the other, and the signal characteristic, for example scattered light,. of these complexes does not differ.
Such immunochemical determination methods are known per se to the person skilled in the art, for example from EP 0 252 127.
In addition to the possible ambiguity in the signal concentration relationship, a further problem is the determination of low concentrations and the evaluation of reactions which take place slowly. The reaction profile of the antigen-antibody bonding in principle has a lag-phase at the start, a region where the rate of reaction is a maximum and a saturation region (FIG. 1a-1c). The extent to which these three phases are pronounced is very heavily dependent on the concentration of the. antigen of the antibody and, furthermore, on a large number of other factors, such as the temperature and the dilution medium, although these are kept as constant as possible in a test system.
The present invention was thus based on the technical problem of providing an immunochemical determination method with the aid of light scatter produced by specific antibodies, which method not only allows very low concentrations to be measured but also offers a high level of protection against the "high-dose hook" effect.
This technical problem is solved by the provision of the embodiments described in the claims.
The essential part of the method according to the invention is that the measurement time window of the respective reaction is adapted by suitable technical steps such that the evaluation takes place reliably in the linear region and the region of the maximum rate of reaction of the time-dependent reaction. The result of such evaluation is V.sub.MaxLin which sometimes is also called X.sub.lin.
The method according to the invention can be ensured by various technical embodiments.
Analytes for the purposes of the invention are plasma proteins such as Ferritin, PSA, IgA, IgG and proteins which can be ascribed to the field of coagulation, such as D-Dimer and clotting factors, in particular genetic variants of clotting factors and, furthermore, haptenes such as hormone and messenger peptide.
The method according to the invention can also advantageously be used for determining the functionality of the clotting system, such as quick test and aPTT that is, an activated partial thromboplastin time.
It has thus surprisingly been found that the determination method described in the following text not only allows the measurement of low concentrations but also ensures increased protection against the "high-dose hook" effect.